Identification of autophagy-associated circRNAs in sepsis-induced cardiomyopathy of mice

Circular RNAs (circRNAs) play a role in sepsis-related autophagy. However, the role of circRNAs in autophagy after sepsis-induced cardiomyopathy (SICM) is unknown, so we explored the circRNA expression profiles associated with autophagy in an acute sepsis mouse model. At a dose of 10 mg/kg, mice were intraperitoneally administered with lipopolysaccharides. The myocardial tissue was harvested after 6 h for microarray analysis, qRT-PCR, and western blotting. Gene Ontology, Kyoto Encyclopedia of Genes and Genomes and Gene Set Enrichment Analysis were evaluated, and a competing endogenous RNA network was constructed, to evaluate the role of circRNAs related to autophagy in SICM. In total, 1,735 differently expressed circRNAs were identified in the LPS-treated group, including 990 upregulated and 745 downregulated circRNAs. The expression level of the autophagy-specific protein p62 decreased, while the ratio of LC3 II to LC3 I increased. Additionally, 309 mRNAs and 187 circRNAs were correlated with autophagy in myocardial tissue after SICM. Of these, 179 circRNAs were predicted to function as “miRNA sponges”. Some distinctive circRNAs and mRNAs found by ceRNA analysis might be involved in autophagy in SICM. These findings provide insights into circRNAs and identified new research targets that may be used to further explore the pathogenesis of SICM.

Gene set enrichment analysis (GSEA). GSEA was performed to identify the markedly enriched gene set clusters in myocardial tissue. The enrichment score (ES) curve was built using the GSEA4.3.1 software. The gene set with False Discovery Rate (FDR) < 0.25, |normalized enrichment scores (NES)|> 1, and nominal (NOM) p value < 0.05 was recognized as statistically significant.
Statistical analysis. All data were shown as mean ± standard deviation. GraphPad Prism 9.0 was used for analysis. Normality test was performed by using Shapiro-Wilk test. Student's t test was used to compare significance between two groups. P < 0.05 was considered statistically significant.

Results
CircRNA expression profiles in myocardium of septic mice. After LPS injection, mouse hearts displayed an increased MDA content and a decreased SOD activity ( Fig S1). Meanwhile, the mitochondrion bloated and bubbled, and cristae was disrupted in the mice myocardial treated with LPS (Fig. S1). All the above results indicated that the animal model of sepsis-induced cardiomyopathy was established. We performed RNA sequencing on mouse hearts (Fig. 1A). The distribution of circRNA expression profiles in all samples showed good symmetry and dispersion (Fig. 1B). Differentially expressed circRNAs (DE circRNAs) were illustrated in red or blue color (Fig. 1C, D). Red color represents twofold upregulation of circRNAs, while blue color represents twofold downregulation of circRNAs in Fig. 1C. In Fig. 1D, circRNAs with fold change ≥ 2 (p < 0.05) and those with fold change ≤ 0.05 (p < 0.05) are shown in red or blue color, respectively. The expression features of dysregulated circRNAs were evaluated (Fig. 1E). In the LPS group vis-à-vis the control group, 1,735 circRNAs were differentially expressed (fold change > 2, p < 0.05), including 990 upregulated (57.06%) and 745 downregulated (42.94%) circRNAs. Depending on the degree of the fold change, the top 20 DE circRNAs are listed in Table 1. The genomic locations of the 1735 dysregulated circRNAs transcribed from all chromosomes, except chromosomes X and Y, are shown in Fig. 2A.  DE CircRNA function analysis. The potential functions of 1735 DE circRNAs were predicted by GO and KEGG enrichment analyses, and the results are shown in bubble charts ( Fig. 3A-B). Based on the size of the enriched factors, the top 30 most remarkably enriched GO items were selected (Fig. 3A). The results showed that the host genes of DE circRNAs during LPS treatment were mostly involved in the "negative regulation of metalloenzyme activity" and "I-kappa B/NF-kappa B complex" (Fig. 3A). The KEGG pathway for enrichment   CeRNA network prediction and annotation. The functions of circRNA include competitive adsorption of microRNAs (miRNA), regulation of RNA-binding proteins, and modulation of variable cleavage or transcription. CircRNAs bind to the corresponding miRNAs by MREs, which act as "sponges" preventing miRNA binding to the target gene and then jointly participating in the expression regulation of the target genes. This mechanism of action is called the "ceRNA mechanism". This is the main research concept related to circRNAs. We filtered the DE circRNAs shown in Table 1 and found four associated mRNAs using ceRNA analysis: cir-cRNA.2982, circRNA.2983, circRNA.2986, and mmu_circ_0006655. The result is shown in Fig. 4 and shows that the ceRNA mechanism exists after SICM.
First, to confirm the protein expression of autophagy markers in mouse myocardium after SICM, we performed western blotting to detect the levels of proteins p62 and LC3 (Fig. 5A). Compared to the control group, the LPS group saw a decrease in the expression of p62 protein (1.80 ± 0.12, p < 0.05, Fig. 5B) as well as a significant increase in the ratio of LC3 II to LC3 I (1.60 ± 0.38, p < 0.01, Fig. 5C), suggesting the occurrence of autophagy. GSEA indicated that the gene set related to positive regulation of autophagy was enriched in SICM damage (Fig. 5D). The results further confirmed that autophagy occurs in mouse cardiomyocytes after 6 h of LPS treatment. In the animal autophagy signaling pathway (KEGG: mmu04140), several circRNA-related host genes were altered after SICM, with some of them (e.g., REDD1, FLIP, Bcl-XL, and TBK1) upregulated and others (e.g., RAB7B and RUBCN) downregulated (Fig. 5E). Next, we predicted the pathways associated with autophagy via KEGG pathway analysis and found nine autophagy-associated endogenous signaling pathways (Fig. 7A-B).

Discussion
In the present study, a mouse model of sepsis-induced cardiomyopathy was established. A total of 1735 differently expressed circRNAs were identified in the LPS-treated mouse hearts. And 187 circRNAs were found to be related to 309 autophagy-associated mRNAs in septic myocardial tissue. Among these, 179 circRNAs were predicted to function as "miRNA sponges". There is a high mortality rate of SICM 2 . Dysregulated autophagy is one of the main pathophysiological events in SICM 25,26 . Some studies have found that circRNAs, such as circCDYL, circCUL2, circRNA_002581, and circEXOC5, were involved in regulating autophagy, even in an LPS-challenged mouse cell model 18,[27][28][29] . The abovementioned reports suggest a role of circRNAs in sepsis-induced autophagy. In our study, we established an SICM mouse model and confirmed the occurrence of the autophagic response in the mouse myocardium tissue by testing the protein levels and performing GSEA for LC3 and p62. In total, 1735 dysregulated circRNAs (including 990 upregulated and 745 downregulated circRNAs) were found in septic mice myocardium tissues using microarrays. In these DE circRNAs, 187 circRNAs are related to autophagy on GO and KEGG pathway analyses.
Autophagy is a fundamental cell protection pathway, and lysosomes are the site of intracellular autophagy [30][31][32] . As autophagy-marker proteins, LC3 and p62 are related to autophagosomal membrane formation 33 . In the present study, the p62 protein expression decreased, while the ratio of LC3 II to LC3 I increased after LPS treatment  www.nature.com/scientificreports/ (vs. control group, p < 0.05). This is consistent with the findings from previous studies 34,35 . Although autophagy was not present in the top 30 pathways shown in Fig. 3A, B, we still found that the gene set related to "positive regulation of autophagy" was enriched in the LPS group by GSEA. Several molecular mechanisms may participate in autophagy. In our study, we found nine autophagy-associated endogenous signaling pathways based on KEGG pathway analysis. These signaling pathways associated with autophagy are involved in various cellular and animal models [36][37][38][39] . For example, ER stress-induced autophagy, which was mediated by oxidative stress, decreased via the modulation of the PI3K-related cascade reaction in acute lung injury in LPS-induced mice 40 . In an ischemic/reperfusion-induced H9C2 cell injury model, autophagy induced by HIF-1α/BNIP3 signaling pathway protects the myocardium 41 . Laminar flow-induced endothelial autophagy and SIRT1 expression due to inhibited Hippo/YAP signaling pathways interrupt atherosclerotic plaque formation 42 . Notably, the HIF-1 and Hippo signaling pathways are involved in autophagy regulation, but the regulatory role has not been adequately explored in SICM-induced autophagy.
The role of circRNAs in SICM-induced autophagy has not been fully explored. Additionally, circRNAs that act as "sponges" are involved in the regulation of target gene expression. Depending on GO and KEGG analyses, we found 179 autophagy-related circRNAs that may bind to miRNAs. Autophagy-associated mRNA-binding sites also exist on the same miRNAs. Thus, we predicted the SICM-induced autophagy-related target genes using the ceRNA networks. For example, circRNA.27393 showed the top fold-change among the autophagy-related circRNAs and might regulate the mRNAs, such as the death-associated protein (DAP), ring-finger protein 152 (rnf152), and Ajuba by sponging mmu-miR-1933-3p, mmu-miR-448-5p, mmu-miR-125a-5p, and mmu-miR-125b-5p. These predicted mRNAs are associated with autophagy [43][44][45] . Other predicted autophagy-related mRNAs identified on ceRNA analysis in our research have also been proved to be related to autophagy, such as Bnip3, PPP2R2A, eEF2K, and IGF1 46-49 . www.nature.com/scientificreports/ In our previous research, we identified the mitochondrial function-associated lncRNAs in SICM 19 . Bnip3 and PPP2R2A are predicted to be autophagy-related mRNAs and are associated with mitophagy 46,47,50 . These results showed that circRNAs and lncRNAs might regulate mitochondrial function and degradation after SICM. Ajuba Tead3, Serpine1, Gli2, and Bmp2 were predicted to be related to the Hippo signaling pathway and were involved in autophagy 45,[51][52][53][54] . Serpine1 and Bcl2 were predicted to be related to the HIF signaling pathway and autophagy 51,55 . Therefore, our results provide new ideas to further evaluate the role of the HIF-1 and Hippo signaling pathways in LPS-induced cardiomyocyte autophagy. The role of certain predicted circRNAs and mRNAs in SICM-induced autophagy has not been elucidated and should be evaluated in future studies.
Sepsis is a life-threatening organ dysfunction. Autophagy is a major pathogenesis of sepsis-induced cardiomyopathy. Our study identified differently expressed circRNAs in the hearts of septic mice. We also gained some specific circRNAs and their potential target mRNAs which might be involved in autophagy in septic hearts. These  www.nature.com/scientificreports/ findings offer a fine view of circRNAs and might allow developing new treatment strategies for sepsis-induced cardiomyopathy and reducing the incidence and mortality of sepsis. Because of the limitations of our detection methods, our experiment also has some limitations. First, the present study only focused on the acute phase of sepsis. Expression profiles of cirRNAs associated with autophagy in the chronic phase of sepsis needs to be further explored, which might provide a more panoramic view of autophagy-related circRNAs in sepsis-induced cardiomyopathy. Second, the combination of various methods is more reliable to detect circRNA, such as PCR, RNase R, and Northern blot. We explored the circRNAs using only qRT-PCR 56,57 . Third, autophagy-related pathways were not studied in detail, and we only screened circRNAs based on the reported autophagy pathways. Therefore, some potentially undiscovered circRNAs may be missed. Fourth, although we identified some potential autophagy-related circRNAs (such as circRNA.27393, circRNA.27392, and circRNA.5564), the mechanism of action of these circRNAs in SICM needs further in vivo and in vitro studies.

Conclusions
Our data indicate that the circRNAs, including circRNA.27393, may influence SICM-induced autophagy. Our research provides a new potential treatment strategy for SICM via the regulation of autophagy by circRNAs.

Data availability
The microarray data of circRNAs and mRNAs have been deposited in the GEO database (GSE142615).